Glycan determinants synthesized by specific glycosyltransferases play defined roles in development, cancer progression, metastasis, immunity and Notch signal transduction. In order to understand new and conserved functions of sugars in biology, the repertoire of glycan structures synthesized by model organisms (the glycome) must be defined. Genes that encode glycosyltransferases that produce the glycome must be isolated and the biochemical activities of their producs determined. The approximately 50 independent CHO glycosylation mutants that we have characterized under the auspices of this grant provide unique access to glycomes and to functional glycomics. Mutants with an established glycosylation defect have proved extremely useful for expression cloning glycosylation genes, for determining lectin binding specificities, and, most recently, for showing that fringe is a novel glycosyltransferase that directly modifies Notch and thereby inhibits Jagged-1-induced Notch signaling. We now propose to use CHO cells and the CHO glycosylation mutants to identify the activity of "orphan" glycosyltransferases predicted from genome and mutation database searches. The focus will be on glycosyltransferases that modulate Notch signaling in the CHO co-culture assay and those whose acceptor specificity is highly conserved across species. The corresponding glycosyltransferase genes will be functionally localized in developmental and signaling pathways by characterizing existing mutants or by gene silencing and antisense techniques in Drosophila, C. elegans and/or zebrafish. In a second aim, the number of CHO glycosylation mutants available for experiments in functional glycomics will be expanded by determining the biochemical defect and genetic basis of previously isolated loss- and gain-of-function CHO glycosylation mutants using rapid analytical methods and retrovirus expression cloning. Thirdly, the entire panel of CHO glycosylation mutants and certain glycosyltransferase gene transfectants which present a wide variety of multivalent, differentially galactosylated cell surface environments, will be used to characterize binding and functional interactions of galectins 1, 2, 3 and 4, which represent three distinct structural types of galectin.